Loss of internal structural order induced by freezing and lyophilization correlates with reduced in vitro activity of mRNA lipid nanoparticles

mRNA-loaded lipid nanoparticles (mRNA-LNPs) hold promise for vaccines and therapeutics, yet the relationship between their structure and efficacy remains elusive. Here, we generated 58 mRNA-LNP samples with varied structural properties and in vitro cellular activities by freezing and lyophilization to investigate the structure-activity relationship (SAR). Our findings revealed that the intensity of a characteristic peak, as detected by small angle X-ray scattering (SAXS), demonstrated a strong correlation with in vitro activity while the encapsulation efficiency is high. This observation suggests that a more ordered internal structure is likely associated with enhanced in vitro activity of mRNA-LNPs. Further analysis using ³¹P nuclear magnetic resonance indicated that lyophilization might induce phase separation of mRNA and lipids within the LNPs, leading to a diminished SAXS peak and reduced in vitro activity compared to fresh mRNA-LNPs. Notably, this in vitro SAR was proved context-dependent rather than universal, and cannot be extrapolated to in vivo settings due to species- and route-dependent complexity. Collectively, these findings identify internal structural order as a robust descriptor for optimizing formulation processing and storage, demonstrate that freezing- and lyophilization-induced structural disruption compromises in vitro activity, and support high-throughput SAXS-based screening of LNP libraries to accelerate rational development of stable mRNA nanomedicines.